Loki
Nikon E600 Widefield Microscope
User Guide
v. 1.2 (03/2015)
Objectives
10x/0.3NA Plan Fluor PH1
20x/0.5NA Plan Fluor PH1
40x/0.95NA Plan Apo DIC
(correction collar for coverslip thickness)
40x/0.75 Plan Fluor PH2
100x/1.3NA oil Plan Fluor PH3
Standard coverslip thickness 0.17
(#1.5 coverslip)
Xenon lamp
Brightfield, Dapi, Fitc/GFP, Tritc, Cy5
Cameras
Nikon Digital Sight CCD camera (DS-Qi; 6.45 m x 6.45 m pixel)
& NIS Elements for fluorescence imaging (monochrome)
Spot Insight QE CCD camera (model 4.3; 7.4 m x 7.4 m pixel)
& Spot Basic 5.1 for transmitted light imaging (color)
 NOTE: the hour meter on the light source is broken, so please ensure all use is logged
Contents:
Quick Guide
3
Starting up the System
4
Transmitted Light / Brightfield
5
Kohler Alignment for optimal contrast
For DIC Imaging using the 40x DIC objective
For Phase Contrast Imaging
6
Taking Transmitted Light Images using Spot Basic / Spot Insight Color Camera
8
Reflected Light / Fluorescence
10
When you’re finished
13
APPENDIX A –Saving to your lab’s folder on MCDB Dept Server
14
APPENDIX B – Parts of the microscope
15
Loki User Guide v. 1.2
2
Quick Guide:
1) Turn on components in order.
2) Log into Windows with your IdentiKey and start appropriate software.
3) Put sample on scope coverslip up. If using oil immersion, do not switch back to air
objective. No oil on air objectives!
4) Find and focus your sample through the oculars.
5) Acquire images, timelapses, Z-series, etc.
6) Save and copy your data. The LMCF is not responsible for long-term data storage. See
Appendix A for information on how to transfer data to MCDB Dept Server.
7) Rotate objective out of the way and remove sample from stage. Properly clean oil off of
any immersion objectives you may have used with lens paper. If you are at all unsure of
this process, ask for help!
8) Leave the microscope on a low power objective for next user.
9) Close software.
10) Log use in Excel sheet, save and close.
 If someone is signed up within the next two hours (Check the MCDBCal!):
11) Log off Windows and leave components on.
 If no one is signed up within the next two hours or you are the last of the day:
11) Shut down system in reverse numerical order.
12) Cover the microscope.
Loki User Guide v. 1.2
3
Starting Up the System:
Non-optional steps are highlighted yellow.
For fluorescence imaging only, complete blue steps.
For transmitted light imaging only, complete green steps.
1) Turn on the power bar labelled “Loki” (switch is on the side near the
lights)
2) Turn on the xenon lamp if you will be imaging fluorescence. If the
xenon lamp was turned off within the last 30 minutes – WAIT to let
the lamp cool before turning it back on. You must turn on the xenon
lamp before powering on the other components.
3) Turn on the microscope base if you will be imaging transmitted light.
4) Turn on the automated turret controller power.
5) Turn on the Spot Insight QE camera if you will be imaging transmitted
light.
6) Turn on the Nikon Digital Sight controller if you will be imaging
fluorescence.
7) Turn on the Prior OptiScan automated focus controller.
8) Turn on the PC.
Loki User Guide v. 1.2
4
Transmitted Light / Brightfield:
1) Make sure the microscope base (#3) is on since this powers the
transmitted light.
2) Manually rotate to a low power objective and place sample on
stage, coverslip up.
3) Using the remote on the right, set the dichroic position to
1 or 6 (these are empty positions). To change cubes click
the cube buttons until 1 or 6 has a little green light.
4) Ensure that the manual slider is pushed all the way in to
send the light to the oculars instead of the camera.
5) Using the Prior controller, adjust the focus to find your
sample. Please do not turn the focus knob on the
microscope itself as this can damage the Prior motor.
6) Adjustment of the light intensity can be done using the
lamp adjustment knob (not instantaneous) or inserting a neutral
density filter into the light path.
Lamp
adjustment
knob
Loki User Guide v. 1.2
Neutral
density (ND)
filters
5
Kohler Alignment for optimal contrast:
These steps will need to be performed for each objective.
1) Focus your sample using the Prior controller. Select an empty position on the condenser
wheel (“A”). Ensure the DIC polarizer and analyzer are out of the light path (for
locations, see image on next page).
2) Move the condenser lens up close to the lower surface of the slide, and open the
condenser diaphragm all the way.
3) Close the field diaphragm and bring its edges into focus using the condenser height
adjustment knob.
4) Use the x-y translation screws to center the image of the closed-down field diaphragm.
5) Once the field diaphragm is focused and centered, open it back to where it just leaves
the edges of the field of view.
6) Now you need to adjust the condenser diaphragm. If it is closed down too much, your
image will appear dark and degrade resolution. If it is opened too much, you will lose
contrast. To view the aperture diaphragm superimposed over the objective rear focal
plane, remove the left eyepiece. Adjust the size of the condenser diaphragm so that it
illuminates 65-80% of the objective back focal plane. Reinsert the eyepiece.
Condenser height adjustment
X-Y translation screws
Condenser
diaphragm
Field diaphragm
Loki User Guide v. 1.2
6
For DIC Imaging using the 40x DIC objective:
1) After Kohler alignment with 40x DIC objective, insert the polarizer by rotating it into
place above the field diaphragm and insert the analyzer.
2) Rotate the condenser turret to DIC M to match the objective.
3) Adjust polarization angle to achieve desired level of contrast.
Analyzer
Polarizer
For Phase Contrast Imaging:
1) After Kohler alignment with desired objective,
rotate the condenser turret to the appropriate
phase annulus (PH1, PH2, PH3) depending on
the objective.
2) Remove the left ocular and make sure that the
condenser phase annulus and objective phase
plate are properly aligned using the condenser
X-Y translation screws and you see a fully
illuminated ring. Reinsert eyepiece.
http://www.microscopyu.com/tutorials/java/phasecontrast/microscopealignment/
Loki User Guide v. 1.2
7
Taking Transmitted Light Images using Spot Color Camera:
1) After you have focused and aligned your sample as desired, open the Spot
software.
2) Pull out the lower knob to send the light to the cameras and
pull out the upper knob to send the light to the Spot camera
(instead of the Nikon fluorescence camera).
3) Open the Live and Image Settings windows.
4) Go to an empty region of your slide and click “White balance” to show the software
what should be white.
Loki User Guide v. 1.2
8
5) At any point you can use the controls at the bottom of the
live window to Pause the live streaming, Close the window, or
Restart to recompute the autoexposure time. This can also
be done on the “Exposure” tab of the Image Settings window
(button on the bottom labeled “Recompute Exposure”).
6) The recommended starting settings for transmitted light
image acquisition are: Auto Exposure, Brightfield-transmitted
light, Gain of 1, and 1 sec Live Image Maximum Exposure. In
the Area tab, it is recommended that you just use Full Chip
without rotation.
7) Now adjust the image to capture desired features. You can adjust Brightness on the
Exposure tab, Saturation on the Appearance tab (makes colors look deeper, so light blue
becomes more pronounced), Gamma on the Appearance tab (increasing gamma will
make dark regions darker and decreasing gamma will make dark regions lighter),
Contrast on the Appearance tab (adjusting the difference between light and dark
regions), and Color Temperature (down makes the image “warmer” more yellow/orange
and up makes the image “colder” or more blue).
8) Once image looks good, click Acquire a single image button at the top.
9) Save the image in the C:\SAVE YOUR DATA HERE\{your lab} folder. It is recommended
that you save the image as a TIFF.
Loki User Guide v. 1.2
9
Taking Fluorescence Images using NIS Elements / Nikon DS-Qi Camera:
1) Open NIS Elements BASIC software.
2) Using Prior focus knob and filter controller, find and focus on your sample.
Ensure slider is pushed in for BINO (light to eyepieces), analyzer is out of the
light path, ND filters are out of the light path for maximal fluorescence signal.
3) Switch to camera acquisition by pulling out the lower knob (to the PHOTO position) and
pushing in the camera selection knob (to NIKON position).
4) Along the top bar of NIS Elements, choose the appropriate optical configuration
shortcut (Dapi, FITC, TRITC, or Cy5) and click the Live Quality button (looks like a play
button with a camera). This will show you what the image looks like so you can adjust
the focal position with the Prior controller. You can change which filter is in position
using the shortcuts along the top or use the remote control. Click the EPI button to
open or close the fluorescence shutter.
Loki User Guide v. 1.2
10
5) Once in the correct focal plane, you
can adjust the exposure time and
analog gain on the right. These are
adjusted for each filter position. If
you’re happy with the appearance of
the image, you can take a single plane
image using the Snap button (camera
symbol) at the top toolbar. Make
sure you are not
overexposing/saturating the image by
looking at the lookup table (LUT)
image on the right-hand side – make
sure it does not reach max value.
6) Adjust settings for each color you
want to image.
7) To take a Z-series, timelapse, and/or multiple colors, open the ND Acquisition window
(docked window on right, or use ND shortcut button on top, or green ND button on
right). You cannot do XY positions on this microscope (no motorized XY stage).
8) Check on the appropriate tabs for your experiment – you can pick 2 max. On the
Lambda tab, choose the optical configurations you want to image. The Z series tab will
allow you to set the range to image either by setting the top and bottom or selecting a
range around the current position. To range around your current position, click the
home button (to set this as the center) and choose the appropriate step size / number
of sets / total range to image (you need to choose two of three). On the Time tab,
choose the interval length and total time to image (remember that your interval length
can be determined by the total time it takes to acquire the z planes at each timepoint).
Loki User Guide v. 1.2
11
9) Uncheck the save to file option.
10) When you’ve selected everything, click run now. Wait. The progress window will show
you how many z positions and/or time positions are left.
11) When done acquiring images, save the .nd2 file in the SAVE YOUR DATA HERE folder.
12) To save your images as a TIFF series, go to File  Import/Export  Export ND
Document.
Loki User Guide v. 1.2
12
When you’re finished:
1) Save and copy your data. The LMCF is not responsible for long-term data storage. See
Appendix A for information on how to transfer data to MCDB Dept Server.
2) Rotate objectives away from sample and remove sample from stage. Properly clean oil
off of any immersion objectives you may have used with lens paper. If you are at all
unsure of this process, ask for help!
3) Close software.
4) Log use in Excel sheet, save and close.
 If someone is signed up within the next two hours (Check the MCDBCal!):
5) Log off Windows and leave components on.
 If no one is signed up within the next two hours or you are the last of the day:
6) Shut down system in reverse numerical order.
7) Cover the microscope.
Loki User Guide v. 1.2
13
APPENDIX A – Saving to your lab’s folder on Collie (MCDB Dept Server):
 Questions regarding MCDB Server should be directed to Erik Hedl.
1) Map the network drive. Right click on Computer icon on desktop or Computer in Start
menu. Click on “Map network drive…”
2) Drive letter should be Z: and the folder should be \\collie.int.colorado.edu\<your lab
name> (for example \\collie.int.colorado.edu\OlwinLab).
If your login for Collie is not your IdentiKey, check the box “connect using different
credentials.”
3) Click finish.
4) If you selected “connect using different credentials,” enter your username and
password.
5) Click Ok and the drive should now appear. Copy over your files. If you have a lot of files,
be sure to allow time for the transfer. Under ideal conditions you will be able to copy
close to 42 gigabytes per hour, don’t count on ideal conditions. Also, it is safer to copy
(not move) your files, then delete after the copy has completed.
 You cannot, must not, should not reserve time on the microscopes in MCDB Cal to copy
data – reservations are for imaging only.
 The LMCF PCs are not long-term data storage places and we are not responsible for lost
data.
 Do not forget to always safely store and backup your raw data – this represents the
ground truth of what you acquired that day and has all the associated metadata. Many
journals are now requiring that you submit raw data with your manuscript.
Loki User Guide v. 1.2
14
APPENDIX B – Parts of the
microscope:
(not identical to our microscope; image
is lacking automated dichroic turret)
Loki User Guide v. 1.2
15